The present invention relates to the preparation of submucosa-derived gel compositions and their use for inducing the proliferation and growth of cells in vivo and in vitro. More particularly, this invention is directed to an improved matrix comprising warm-blooded vertebrate submucosa that has been fluidized by enzymatic digestion and then gelled to form a shape retaining matrix. In one embodiment the matrix is used as an improved cell culture substrate to support the growth and tissue differentiation of eukaryotic cells in vitro. Alternatively, the compositions of the present invention can be implanted or injected into a host to induce cell growth and proliferation in vivo.
Tissue culture allows the study in vitro of animal cell behavior in an investigator-controlled physicochemical environment. However, cellular morphology and metabolic activity of cultured cells are affected by the composition and architecture of the substrate on which they are grown. Presumably cultured cells function best (i.e. proliferate and/or perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment.
The interaction of cells with their extracellular matrix (ECM) in both in vivo and in vitro environments plays a crucial role in the organization, homeostasis, and function of all tissues and organs. Continuous communication between cells and the surrounding matrix environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, the development of form (morphogenesis), angiogenesis, wound healing, and even tumor metastasis. The cell and its ECM are said to exist in a state of xe2x80x9cdynamic reciprocityxe2x80x9d. Both biochemical and biophysical signals from the ECM modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death.
In turn, the cell can modify its ECM environment by modulating synthesis and degradation of specific matrix components. The realization of the significance of cell-ECM communication has led to a renewed interest in characterizing ECM constituents and the basic mechanisms of cell-ECM interaction.
Currently, studies conducted in vitro for analyzing cellular function are limited by the availability of cell growth substrates that present the appropriate physiological environment for proliferation and function/growth development of the cultured cells. To provide an in vitro cell culture environment which would more closely mimic cell-ECM interaction in vivo, purified ECM components such as collagen, fibronectin, laminin, glycosaminoglycans (e.g., hyaluronic acid, heparan sulfate) have been used to prepare artificial substrata for augmentation of cell adhesion, growth, and morphology. Three-dimensional (3D) culture matrices also have been fashioned from purified ECM components, specifically fibrin clots and collagen gels. Investigations with these matrices have demonstrated the importance of 3D architecture in the establishment of a tissue-like histology.
Complex scaffolds representing combinations of ECM components in a natural or processed form are commercially available. For example, Becton Dickinson currently offers two such products: Human Extracellular Matrix, and MATRIGEL(copyright) Basement Membrane Matrix. Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, H K et al., U.S. Pat. No.4,829,000 (1989).) MATRIGEL(copyright) is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these basement membrane extracellular matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high.
Additional basement membrane matrices utilized as cell culture substrates include allogeneic and xenogeneic compositions prepared from lens capsule, liver, amnion, and chorioallantoic membranes. Although these substrata allow the study of cell growth and differentiation in a more physiologically relevant system, their use has been limited by availability and amenability to disinfection, sterilization, and manufacturing processes.
The present invention is directed to the preparation of a collagenous gel matrix derived from the interstitial extracellular matrix of warm-blooded vertebrate tissues. The predominant collagen types present such matrices are collagen I,III and V. The matrices for use in accordance with the present invention are derived from tissues comprising highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration. One extracellular collagenous matrix for use in this invention is derived from submucosal tissue of a warm-blooded vertebrate.
Submucosal tissue harvested from warm-blooded vertebrates is a collagenous matrix that has shown great promise as a unique graft material for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation and differentiation of cell populations in vitro.
As a tissue graft, submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. Numerous studies have shown that submucosal tissue is capable of inducing host tissue proliferation, remodeling and regeneration of tissue structures following implantation in a number of in vivo microenvironments, including lower urinary tract, body wall, tendon, ligament, bone, cardiovascular tissues and the central nervous system. It has been used successfully in vascular grafts, urinary bladder and hernia repair, replacement and repair of tendons and ligaments, and as a dermal graft. Upon implantation, cellular infiltration and a rapid neovascularization are observed and the submucosa extracellular matrix material is remodeled into host replacement tissue with site-specific structural and functional properties.
Vertebrate submucosa can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. The preparation and use of submucosa as a tissue graft composition is described in U.S. Pat. Nos. 902,508; 5,281,422; 5,275,826; 5,554,389 and other related U.S. patents. Submucosal tissue consists primarily of extracellular matrix material and is prepared by mechanically removing selected portions of the mucosa and the external muscle layers and then lysing resident cells with hypotonic washes. Preliminary biochemical analyses show that the composition of small intestinal submucosa is similar to that of other interstitial extracellular matrix structures, and consists of a complex array of collagen, proteoglycans, glycosaminoglycans, and glycoproteins. The major components commonly identified in extracellular matrix tissues similar to submucosal tissue include growth factors; the cell adhesion proteins, fibronectin, vitronectin, thrombospondin, and laminin; the structural components, collagen and elastin; and the proteoglycans, serglycin, versican, decorin, and perlecan.
Submucosa tissue can be used as a tissue graft construct, or as a cell culture substrate/supplement, in either its native solid form, as a fluidized comminuted form, or as an enzyme digested solubilized form. Furthermore, the solubilized forms of vertebrate submucosa can be gelled to form a semi-solid composition that can be implanted as a tissue graft construct or utilized as a cell culture substrate. As a tissue graft, the enzyme-digested, solubilized form can be injected or otherwise delivered to living tissues to augment, enhance or suppress the structure or function of said tissue. Furthermore, said enzyme-digested, solubilized form can be combined or modified with specific proteins, growth factors, drugs, plasmids, vectors, or other therapeutics agents for controlling the desired augmentation, enhancement or suppression of the recipient tissue function. Still further, said enzyme-digested, solubilized form can be combined with living primary or cultured cells prior to delivery to the living tissues, such combination providing further augmentation, enhancement of suppression of tissue structure or function. The submucosa gel form can also be used as a cell growth substrate, providing a relatively inexpensive cell culture growth substrate that promotes or induces growth and differentiation of cells cultured in vitro.
The present invention is directed to an improved vertebrate submucosa composition comprising a semi-solid translucent interstitial extracellular matrix formed from solubilized submucosa of a warm-blooded vertebrate, and a method of forming that composition. More particularly, the interstitial extracellular matrix comprises submucosa that has been enzymatically digested to form a submucosa hydrolysate, wherein the submucosa hydrolysate is fractionated and then gelled.
The present invention is directed to compositions comprising vertebrate submucosa in gelled form and a method of making an improved submucosa gel. As used herein, a gel is a fluid having a viscosity of greater than about 100,000 cps at 25xc2x0 C., and more typically having a viscosity of about 200,000 to about 350,000 cps at 25xc2x0 C., such that the fluid is in a semi-solid form that only gradually yields to forces that change its form. Gelled forms of vertebrate submucosa can be prepared by increasing the viscosity of solubilized submucosa, and in one preferred embodiment the solubilized submucosa is gelled by inducing the self assembly of the polymer components of the submucosa. In accordance with one embodiment a submucosa gel is prepared by enzymatically treating vertebrate submucosa to produce a submucosa hydrolysate, wherein the submucosa hydrolysate is gelled by raising the pH to about 6.0 to about 7.4. The term xe2x80x9csubmucosa hydrolysatexe2x80x9d as used herein refers to isolated warm-blooded vertebrate submucosa that has been enzymatically treated to reduce the molecular weight of at least some of the submucosa structural components and produce a composition comprising solubilized components of the isolated submucosa. The submucosa hydrolysate may include insoluble and/or nonhydrolyzed components of the isolated submucosa as well as solubilized components.
In accordance with one embodiment of the present invention, an improved method of forming a gel composition comprising vertebrate submucosa is described. The method produces a translucent, sliceable, shape retaining gel, comprising warm-blooded vertebrate submucosa that has been hydrolyzed and fractionated. The term xe2x80x9cshape retaining gelxe2x80x9d is defined herein to refer to a gel that holds its three dimensional molded shape (i.e. no significant change in the height, length or width) in a hydrated environment for at least one hour at 20xc2x0 C. after removal from the mold and placement on a flat surface without any other support. The method of forming the shape retaining gel of the present invention comprises the steps of enzymatically treating warm-blooded vertebrate submucosa to produce a hydrolysate of vertebrate submucosa having multiple hydrolyzed submucosa components, fractionating the hydrolysate to remove at least a portion of the hydrolysate components and gelling the fractionated hydrolysate. Advantageously, the present method enables the formation of a translucent, shape retaining gel from a complex extracellular matrix without purification of the matrix collagen compounds. Accordingly, the submucosa gel retains many of the original components of the solid delaminated vertebrate submucosa. Furthermore, the gel compositions are particularly well suited for use as cell culture substrates since their relative transparency allows for direct visualization of cells growing on and/or within the submucosa gel matrix.
Submucosal tissue used as the source and starting material for the gel compositions of the present invention comprises submucosa isolated from warm-blooded intestinal tissue as well as other tissue sources such as the alimentary, respiratory, urinary or genital tracts of warm-blooded vertebrates. The preparation of intestinal submucosa is described and claimed in U.S. Pat. No. 4,902,508, the disclosure of which is expressly incorporated herein by reference. Urinary bladder submucosa and its preparation is described in U.S. Pat. No. 5,554,389, the disclosure of which is expressly incorporated herein by reference. Stomach submucosa has also been obtained and characterized using similar tissue processing techniques. Such is described in PCT published application No. WO98/25636, published on Jun. 18, 1998, titled STOMACH SUBMUCOSA DERIVED TISSUE GRAFT, the disclosure of which is expressly incorporated herein by reference. Briefly, stomach submucosa is prepared from a segment of stomach in a procedure similar to the preparation of intestinal submucosa. A segment of stomach tissue is first subjected to abrasion using a longitudinal wiping motion to remove the outer layers (particularly the smooth muscle layers) and the luminal portions of the tunica mucosa layers. The resulting submucosa tissue has a thickness of about 100-200 micrometers, and consists primarily (greater than 98%) of acellular, eosinophilic staining (HandE stain) extracellular matrix material.
Preferred submucosal tissues for use as a source of gelled compositions of the present invention include intestinal submucosa, stomach submucosa, urinary bladder submucosa, and uterine submucosa. Intestinal submucosal tissue is one preferred starting material, and more particularly the tunica submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa of warm-blooded vertebrate intestine. In one embodiment of the present invention the intestinal submucosal tissue comprises the tunica submucosa and basilar portions of the tunica mucosa including the lamina muscularis mucosa and the stratum compactum which layers are known to vary in thickness and in definition dependent on the source vertebrate species.
The isolated vertebrate submucosa is typically rinsed extensively with a hypotonic solution to lyse any cells still associated with the submucosal matrix and to eliminate cell degradation products. To produce the solubilized forms of submucosa utilized to form the gel compositions of the present invention, the submucosa is treated with a disruptive agent that solubilizes the submucosa without substantial destruction of the collagen components of the submucosa. In one embodiment the submucosa is treated with one or more enzymes for a predetermined length of time sufficient to hydrolyze at least a portion of the submucosa structural components and produce a submucosa hydrolysate. Typically the submucosa is comminuted before enzymatic digestion of the submucosa by tearing, cutting, grinding, or shearing the harvested submucosal tissue. More particularly, the submucosa can be comminuted by shearing in a high speed blender, or by grinding the submucosa in a frozen or freeze-dried state, and then lyophilizing the material to produce a powder having particles ranging in size from about 0.1 to about 1.0 mm2. The submucosa powder can thereafter be hydrated with water or buffered saline to form a submucosal fluid of liquid or paste-like consistency. In one preferred embodiment the submucosal tissue is comminuted by freezing and pulverizing the submucosa under liquid nitrogen in an industrial blender. The preparation of fluidized forms of submucosa tissue is described in U.S. Pat. No. 5,275,826, the disclosure of which is expressly incorporated herein by reference.
Enzymatic digestion of the submucosa is conducted under conditions that retain the ability of the endogenous submucosa collagen fibers to self assemble. The concentration of the enzyme used is adjusted based on the specific enzyme used, the amount of submucosa to be digested, the predetermined time of digestion, the temperature of the reaction, and the desired properties of the final product. In one embodiment about 0.1% to about 0.2% of enzyme (pepsin, for example) is added and the digestion is conducted at 4xc2x0 C. for 72 hours. However the digestion can be conducted at any temperature ranging from 4-37xc2x0 C. and the digestion times can be adjusted accordingly from 2-180 hours. In general, the ratio of the concentration of submucosa (hydrated) to total enzyme ranges from about 25 to about 125 and more typically the ratio is about 50, and the digestion is conducted at 4xc2x0 C. for 24-72 hours. The composition of the gel produced from the submucosa hydrolysate will vary, at least in the proportion of their components if not also in the gel contents, depending on the length of digestion and digestive agent used.
The enzymes or other disruptive agents used to solubilize the submucosa should be removed or inactivated before or during the gelling process so as not to compromise gel formation or subsequent gel stability. Also, any disruptive agent, particularly enzymes, that remain present and active during storage of the tissue will change the composition and potentially the gelling characteristics of the solution. Enzymes, such as pepsin, can be inactivated with protease inhibitors, a shift to neutral pH, a drop in temperature below 0xc2x0 C., heat inactivation or through the removal of the enzyme by fractionation. A combination of these methods can be utilized to stop digestion of the submucosa at a predetermined endpoint, for example the submucosa can be immediately frozen and later fractionated to limit the digestion of the submucosa.
The submucosa is enzymatically digested for a sufficient time to produce a hydrolysate of submucosa components. Typically the submucosa is treated with one enzyme, however the submucosa can be treated with a mixture of enzymes to hydrolyze the structural components of the submucosa and prepare a hydrolysate having multiple hydrolyzed submucosa components of reduced molecular weight. The length of digestion time is varied depending on the application, and the digestion can be extended to completely solubilize the submucosa. More preferably the submucosal tissue is partially solubilized to produce a submucosa digest composition comprising hydrolyzed submucosa components and nonhydrolyzed submucosa components.
In one embodiment the digest composition is further manipulated to remove at least some of the nonhydrolyzed components of the submucosa. For example, the nonhydrolyzed components can be separated from the hydrolyzed portions by centrifugation. Alternatively, other separation techniques familiar to those skilled in the art, such as filtration, can be used in accordance with this invention. Accordingly, partially solubilized submucosa can be filtered or subject to centrifugation to remove insoluble portions of the digest composition and thus form a substantially uniform hydrolysate of submucosal tissue. Removal of undigested submucosa from the hydrolysate does alter the composition of the hydrolysate but does not significantly alter the hydrolysate""s ability to form a shape retaining gel.
The conditions used in the digestion of the submucosa produce a hydrolysate having an ionic strength that is not optimal for forming a shape retaining gel. The appropriate ionic strength can be obtained by fractionation of hydrolysate, however, the production of a shape retaining gel from submucosa hydrolysate species is believed to require that those species remain in solution during the fractionation step. Fractionation of the submucosa hydrolysate at physiological pH and physiological ionic strength reduces collagen solubility in the hydrolysate resulting in formation of a weak/non-shape retaining gel. Accordingly, the shape retaining gels of the present invention are prepared from enzymatically digested vertebrate submucosa that has been fractionated under acidic conditions (pH ranging from about 2.0 to less than 7.0). Typically, the submucosa hydrolysate is fractionated by dialysis against a solution having a pH ranging from about 2.0 to about 5.0. In one embodiment, the submucosa hydrolysate is fractionated under mild acidic conditions, wherein xe2x80x9cmild acidic conditionsxe2x80x9d is defined as a pH ranging from greater than 3.0 to less than 7.0. In this embodiment, the submucosa hydrolysate is typically fractionated under mild conditions by dialysis against a solution having a pH ranging from greater than 3.0 to about 5.0. In addition to fractionating the hydrolysate under acidic conditions, the submucosa hydrolysate is typically fractionated under conditions of low ionic strength with minimal concentrations of salts such as those usually found in standard buffers such as PBS (i.e. NaCl, KCl, Na2HPO4, or KH2PO4). Such fractionation conditions work to reduce the ionic strength of the submucosa hydrolysate and thereby provide enhanced gel forming characteristics. In sum, the formation of the shape retaining gels of the present invention is optimized by fractionating the submucosa hydrolysate under acidic conditions and relatively low ionic strength.
The hydrolysate solution produced by enzymatic digestion of the submucosa has a characteristic ratio of protein to carbohydrate. The ratio of protein to carbohydrate in the hydrolysate is determined by the enzyme utilized in the digestion step and by the duration of the digestion. The ratio may be similar to or may be substantially different from the protein to carbohydrate ratio of the undigested submucosal tissue. In accordance with the present invention the submucosa hydrolysate is fractionated under acidic and low ionic strength conditions to remove at least some of the original hydrolysate components. This step produces a fractionated submucosa hydrolysate that has an altered protein to carbohydrate ratio relative to the protein to carbohydrate ratio of the original delaminated submucosa. For example, digestion of vertebrate submucosa with a protease such as pepsin, followed by dialysis will form a fractionated submucosa hydrolysate having a lower protein to carbohydrate ratio relative to the original delaminated submucosa.
In accordance with one embodiment, a shape retaining gel form of submucosa is prepared from delaminated submucosa (having a predetermined protein to carbohydrate ratio) that has been enzymatically digested and fractionated under acidic conditions to form a submucosa hydrolysate that has a protein to carbohydrate ratio different than that of the original delaminated submucosa. In accordance with one embodiment, the submucosa hydrolysate (with or without the nonhydrolyzed submucosa portion) is fractionated by dialysis. The molecular weight cut off of the submucosa components to be included in the gel is selected based on the desired properties of the gel. Typically the pore size will range from about 3,500 to about 10,000, and more preferably from about 3,500 to about 5,000. The hydrolysate is dialyzed against an acidic solution having low ionic strength. For example, the hydrolysate can be dialyzed against a 0.01 M acetic acid (pH of approximately 3.3-3.5). In addition, the submucosa hydrolysate can be optionally sterilized during dialysis by the inclusion of chloroform in the dialysis buffer.
Vertebrate submucosa can be stored frozen (at about xe2x88x9220 to about xe2x88x9280xc2x0 C.) in either its solid, comminuted or enzymatically digested forms prior to formation of the gel compositions of the present invention or the material can be stored after being hydrolyzed and fractionated. Storage temperatures are selected to stabilize matrix components and typically the fractionated submucosa hydrolysate is stored at 4xc2x0 C. for about a week, but it can be stored at 0-4xc2x0 C. for 1-26 weeks, or for longer, if the storage temperature is less than 0xc2x0 C. Submucosa is stored in solvents that maintain the collagen in its native form and solubility. For example, one suitable storage solvent is 0.01 M acetic acid, however other acids can be substituted, such as 0.01 N HCl. In accordance with one embodiment the fractionated submucosa hydrolysate is dried (by lyophilization, for example) and stored in a dehydrated/lyophilized state. The dried form can be rehydrated and gelled to form the shape retaining gel of the present invention.
In accordance with one embodiment, the fractionated submucosa hydrolysate is gelled by adjusting the pH to about 5.0 to about 9.0, more preferably about 6.6 to about 7.4 and typically about 7.0 to about 7.2 thus inducing fibrillogenesis and matrix gel assembly. In one embodiment the pH of the fractionated hydrolysate is adjusted by the addition of a buffer that does not leave a toxic residue, and has a physiological ion concentration and the capacity to hold physiological pH. Examples of suitable buffers include PBS, HEPES, and DMEM. In one embodiment the pH of the fractionated submucosa hydrolysate is first raised to greater than 8.0 by the addition of a base, such as NaOH and then lowered to about 6.0 to about 8.0, more preferably about 6.6 to about 7.4 by the addition of an acid, such as HCl. In accordance with one embodiment, the submucosa hydrolysate is mixed with 10xc3x97PBS Buffer in an 8:1.2 ratio and sufficient 0.05 N NaOH is added to shift the pH to  greater than 8. Then sufficient 0.04 N HCl is added to adjust the pH to between 6.6 and 7.4. The resultant mixture is aliquoted into designated cultureware or appropriate forms and incubated at 37xc2x0 C. for 0.5 to 1.5 hours. The present submucosal gel compositions can be combined with added growth factors, therapeutics, cells, etc., for specific applications (e.g., vehicle for cell delivery, delivery of drugs/therapeutics, 3-dimensional cell culture substrate, and augmentation of tissue repair). The ionic strength of the submucosa hydrolysate is believed to be important in maintaining the fibers of collagen in a state that allows for fibrillogenesis and matrix gel assembly upon neutralization of the hydrolysate. Accordingly, it may be important to reduce the salt concentration of the submucosa enzyme digest prior to neutralization of the hydrolysate.
After the pH of the fractionated submucosa hydrolysate has been adjusted to about 6.0 to about 8.0, more preferably about 6.6 to about 7.4, the solution can be placed in the appropriately shaped container for forming a shaped gel. For example, the solution can be poured onto cell cultureware to conform to the shape of the cultureware before the gel sets. Typically the neutralized, fractionated, hydrolysate is incubated at 37xc2x0 C. to form the gel. The neutralized hydrolysate gels in approximately thirty to ninety minutes at 37xc2x0 C. Alternatively, the gel can be stored at 4xc2x0 C. to delay the setting of the gel for 3-8 hours. The neutralized hydrolysate can be gelled at any temperature ranging from about 4xc2x0 C. to about 40xc2x0 C. Gellation times range from 5 to 120 minutes at the higher gellation temperatures and 1 to 8 hours at the lower gellation temperature. Additional components can be added to the hydrolysate composition before gellation of the composition. For example, proteins carbohydrates, growth factors, bioactive agents, nucleic acids or pharmaceuticals can be added.
The shape retaining gels of the present invention are translucent, having an optical density ranging from about 0.1 to about 2.0 at A405 nm, more preferably from about 0.4 to about 1.2 at A405 nm and more typically about 0.6 to about 0.8 A405 nm. Dialysis of the submucosa hydrolysate against various ionic solutions impacts the turbidity and firmness of the formed gel. The turbidity and firmness of the gel increase relative to the ionic composition of the dialysis solution (PBS less than HClxe2x89xa6Acetic Acid) and is correlated with the matrix component solubility as indicated by a lower initial optical density. Dialysis using a PBS dialysis solution only produced weak gels, whereas dialysis against an acetic acid or HCl solution produces a shape retaining gel having a turbidity of less than 1.2 at A405 nm. After formation of the shape retaining gel, the matrix can be dried/dehydrated and stored. The gel can be subsequently dehydrated without loss of its bioactive properties.
In accordance with one embodiment of the present invention a shape retaining gel matrix is prepared from vertebrate submucosa by enzymatically treating warm-blooded vertebrate submucosa to produce a hydrolysate of warm-blooded vertebrate submucosa. The submucosa hydrolysate is then fractionated to reduce the concentration of hydrolysate components having a molecular weight less than 3500, and the remain fractionated submucosa is gelled by adjusting the pH to about 5.0 to about 9.0, more preferably by adjusting the pH to about 6.0 to about 8.0. In accordance with one embodiment the pH of the fractionated submucosa is adjusted to greater than 8.0 before adjusting the pH to about 6.0 to about 8.0. The method also includes, in one embodiment, the step of separating at least some of the undigested and insoluble components of the submucosa hydrolysate from the solubilized components. One preferred method for removing the nonhydrolyzed components comprises centrifuging the submucosa hydrolysate and recovering the supernatant. Alternatively, the submucosa hydrolysate can be filtered to remove the insoluble submucosa hydrolysate components. The hydrolysate is fractionated to remove at least some of the low molecular weight submucosa hydrolysate species, and typically this step is accomplished by dialyzing against an acidic solution. The pH of the fractionated submucosa hydrolysate is then adjusted to about 6.0 to about 8.0 and the fractionated submucosa hydrolysate is incubated the at 37xc2x0 C. to form the shape retaining gel.
In accordance with one embodiment, a gellable composition is prepared by grinding vertebrate submucosa into a powder and partially digesting the powdered submucosa with 0.1% pepsin in 0.5 M acetic acid for one to two days at 4xc2x0 C. Following partial digestion, the hydrolyzed submucosa is separated from the undigested portions by centrifuging the suspension at 4xc2x0 C. to pellet the undigested material. The supernatant, comprising solubilized submucosa is recovered and the insoluble pellet discarded. The supernatant is then fractionated to remove at least a portion of the hydrolysate components. In one embodiment, the hydrolysate is fractionated by dialyzing the hydrolysate under mild acidic conditions and low salt (i.e., salt concentration lower than physiological salt concentrations). In one embodiment the hydrolysate is dialyzed against several changes of 0.01 M acetic acid at 4xc2x0 C. using dialysis membranes having a molecular weight cut off of 3500. Therefore, in this embodiment the hydrolysate is fractionated to remove the hydrolysate components having a molecular weight of less than 3500. Alternatively, different pore sized dialysis tubing can be used to alter the composition of the submucosa gel formed in accordance with the present invention.
In one embodiment the submucosa is sterilized before formation of the gel, however the submucosa can also be sterilized after the formation of the gel matrix. In one embodiment the submucosa hydrolysate is sterilized during the dialysis step. For example, chloroform (5 ml chloroform per 900 ml of 0.01 M acetic acid) can be added to the dialysis solution to disinfect or sterilize the submucosa. Typically when the submucosa hydrolysate is sterilized by dialysis against chloroform, two additional changes of sterile 0.01 M acetic acid are used to eliminate the chloroform.
In general, isolated vertebrate submucosa can be sterilized using conventional sterilization techniques including glutaraldehyde tanning, formaldehyde tanning at acidic pH, propylene oxide treatment, gas plasma sterilization, gamma radiation, electron beam, peracetic acid sterilization. Sterilization techniques which do not adversely affect the mechanical strength, structure, and biotropic properties of the submucosal tissue is preferred. Preferred sterilization techniques include exposing the submucosa to 1-4 Mrads gamma irradiation (more preferably 1-2.5 Mrads of gamma irradiation) or gas plasma sterilization. Typically, the submucosal tissue is subjected to two or more sterilization processes. After the submucosal tissue is sterilized, for example by chemical treatment, the tissue may be wrapped in a plastic or foil wrap and sterilized again using electron beam or gamma irradiation sterilization techniques.
In accordance with one embodiment a method for inducing the growth of cells in vivo, is provided. The method comprising the step of injecting into a host at a site in need of repair a composition comprising enzymatically digested vertebrate submucosa that is fractionated to reduce the concentration of enzymatically digested vertebrate submucosa components having a molecular weight less than 3500. In one embodiment the fractionated submucosa hydrolysate is neutralized (for example, by adding a physiologically compatible buffer) before injection, and the hydrolysate is injected into the host before the gel matrix sets. The injected material then gels at the in vivo site of injection thus immobilizing the composition at the injection site. The resulting shape retaining gel stimulates endogenous cell proliferation and cell growth/function at the localized injection site and enhances the repair of damaged or diseased tissues. Advantageously this technique allows for the fixation of a matrix composition at a localized site through a minimally invasive procedure. The fractionated hydrolysate can be combined with added growth factors, pharmaceuticals, minerals, bioactive agents or cells prior to injection and formation of the gel matrix.
Alternatively, in one embodiment a shape retaining gel for inducing cell growth in vivo is prepared, comprising a fractionated submucosa hydrolysate in combination with added components. This composition is formed by enzymatically digesting vertebrate submucosa to form a submucosa hydrolysate, and then fractionating the hydrolysate and neutralizing the fractionated hydrolysate to form a shape retaining gel. The additional components are added to the fractionated hydrolysate either before the neutralization step or immediately after the neutralization step and before the gel sets. The mixture is then stirred and allowed to form a shape retaining gel of a predetermined shape. In one embodiment the gel is formed to match the shape of an implantation site in a host and the formed gel is surgically implanted into the host at that site. Various components can be added to the submucosa hydrolysate to form gel matrix compositions in accordance with the present invention, including, but not limited to, proteins, carbohydrates, growth factors, bioactive agents, minerals pharmaceuticals and cells.
The shape retaining gel matrix forms of the present invention can be used as cell culture substrates for supporting the attachment, growth or proliferation of a wide variety of cell types in vitro. The gel matrix comprises a submucosa hydrolysate fraction having multiple hydrolyzed submucosa components, wherein the hydrolysate fraction is prepared from enzymatically digested vertebrate submucosa fractionated to reduce the concentration of hydrolysate components having a molecular weight less than about 3500. The composition is gelled by adjusting the pH to about 6.0 to about 8.0. The gelled forms of submucosal tissue provide a translucent substrate for cell adhesion and also induce cell differentiation. The submucosal tissue is preferably sterilized prior to use in cell culture applications, however nonsterile submucosal tissue can be used if antibiotics are included in the cell culture system. In one embodiment the gelled submucosal tissue is used to coat cultureware (i.e. petri plates, culture bottles or flasks, etc.,) and is used in combination with standard liquid culture media. To prepare gelled submucosa coated cultureware, the fluidized form of submucosal tissue can be poured onto the cultureware and gelled by adjusting the pH of the submucosal tissue to about 6.0 to 8.0.
In accordance with one embodiment a submucosa gel composition is prepared for use in culturing cells in vitro. In one embodiment the composition comprises tissue cultureware that is coated with a composition comprising intestinal submucosa delaminated from both the tunica muscularis and at least the luminal portion of the tunica mucosa, wherein the delaminated submucosa is enzymatically treated, fractionated under acidic conditions to alter the protein to carbohydrate ratio of the original delaminated submucosal tissue, and then gelled. In preferred embodiments the fractionated submucosa hydrolysate is gelled by adjusting the pH of the hydrolysate to about 6.0 to about 7.4. In another embodiment of the present invention, a composition for culturing cells in vitro, comprises tissue cultureware coated with a shape retaining gel matrix comprising an enzyme hydrolysate of warm-blooded vertebrate submucosa that was fractionated to remove at least a portion of the hydrolysate components having a molecular weight less than 3,500, and gelled by adjusting the pH to about 6.0 to about 7.4.
The cell growth substrate of the present invention can be combined with added agents, including minerals, amino acids, sugars, peptides, proteins, glycoproteins, proteoglycans, cytokines, growth factors, drugs, plasmids, vectors, or other bioactive agents that facilitate or inhibit cellular proliferation or differentiation. Other examples of such agents include laminin, fibronectin, epidermal growth factor, platelet-derived growth factor, transforming growth factor beta and fibroblast growth factor. The submucosa substrates of the present invention can be used with commercially available cell culture liquid media (both serum based and serum free). When grown in accordance with this invention, proliferating cells can either be in direct contact with the submucosa or they can simply be in fluid communication with the gelled submucosa. It is anticipated that the cell growth compositions of the present invention can be used to stimulate proliferation of undifferentiated stems cells as well as differentiated cells such as islets of Langerhans, hepatocytes and chondrocytes. Furthermore the described cell growth composition is believed to support the growth of differentiated cells while maintaining the differentiated state of such cells.